An immune system defends against invasion by microorganisms or infiltration by cancer cells by producing antibodies, proteins which can complex with the invading microorganisms or cancer cells and target them for destruction or removal. Any foreign molecule or modified native molecule of sufficient size can act as a stimulus for antibody production. B lymphocytes, the cells that actually produce antibodies, recognize and respond to an antigen by reproducing (cloning) themselves and then producing antibodies specific to that foreign invader (refined antibodies). In viral infections and certain cancers, because the invaders are adept at mutation, there is a competition between the immune response and the virus or cancer cells, with each adapting to mutations made by the other in an effort to achieve victory. A successful outcome for the host requires that the virus or cancer can no longer evade the host defense. This may require a series of antibodies produced by the B cell such that each move and countermove ends in an immunological checkmate, where the “matured” antibody produced is sufficiently refined to ward off all (or most) subsequent attacks from the same or similar invaders. Once the foreign invader is destroyed, the production of the refined antibodies by B cell lymphocytes subsides and the B cell returns to a resting state, although it retains a genetic memory of the foreign invader and can respond effectively on a subsequent attack.
Recent technological advances have made it possible to isolate and cultivate a single clone of lymphocytes to obtain a virtually unlimited supply of antibodies specific to particular disease targets. These antibodies, known as “monoclonal antibodies” because they arise from a single clone of lymphocytes, are produced by hybridoma technology. Conventional and well-established hybridomas technology takes advantage of the fusion (hybridization) of an immortal cell (often a myeloma cell) with the isolated antibody producing cell to produce a hybrid cell known as a hybridomas. For example, in one of the earliest demonstrations of the concept, spleen cells from a mouse presented with an antigen were fused with myeloma cells to create hybridomas. The hybridomas can be isolated by transferring them to a growth fluid that kills off the unfused cancer cells, while the unfused spleen cells die off by themselves.
Hybridomas produce antibodies to the antigen initially injected into the mouse. The single hybridoma or hybridomas secreting antibody into the medium are then screened and those with the desired characteristics are selected. Each hybridoma reproduces itself and these identical self-cloned hybridomas each produce identical monoclonal antibodies having the same antigen affinity (binding strength) and specificity. In this way, a virtually unlimited supply of identical antibodies is created, directed to a highly specific antigen.
The Isolation of B Lymphocytes from Human Blood and the Subsequent Generation of Human Hybridoma Cell Lines have also been Well-Established
To generate immortal cells expressing antibodies useful in therapy of a particular disease, one well-established method is to transform human peripheral blood lymphocytes from a patient with the disease of interest with Epstein Barr virus (EBV) as described for example in Gorny, M. K. et al., Proc. Nat'l. Acad. Sci. USA 86:1624-1628 (1989). Generation of human antibodies from primed donors has also been performed by stimulation with CD40, resulting in expansion of human B cells, Banchreau et al., F. Science (1991), 251:70, Zhang et al., J., Immunol. (1990), 144, 2955-2960, Tohma et al., J. Immunol. (1991), 146:2544-2552, or by an extra in vitro booster step prior to immortalization. Chaudhuri et al., Cancer Supplement (1994), 73, 1098-1104. The B cells resulting from either method are then fused with a myeloma cell, forming immortal monoclonal antibody-producing hybridomas. See, e.g., U.S. Pat. No. 4,897,466 incorporated by reference.